Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

• the invention relates to N-phenyl -2-pyrimidine-amine derivatives in which at least one nitrogen atom carries an oxygen atom to form the corresponding N-oxides, to processes for the preparation thereof, to pharmaceutical compositions comprising those compounds, and to the use thereof in the preparation of pharmaceutical compositions for the therapeutic treatment of warm-blooded animals, including humans.
• the invention relates particularly to compounds of formula I
• R-i is hydrogen or hydroxy
• R 2 is hydrogen, lower alkyl or hydroxy-lower alkyl
• A is -NR 5 R 6 , -CR 5 R 6 or -OR 5 R 6 ,
• R 5 R 6 together represent alkylene with four, five or six carbon atoms, oxa-lower alkylene with one oxygen and three or four carbon atoms, or aza-lower alkylene with one or two nitrogen and two, three or four carbon atoms wherein the nitrogen atom is unsubstituted or substituted by lower alkyl, hydroxy-lower alkyl, or acetyl, and wherein lower alkylene in each case may be partially or totally unsaturated and/or the carbon atoms of lower alkylene may be substituted by lower alkyl, hydroxyl, lower alkoxy or oxo when lower alkylene is not totally unsaturated, and wherein at least one nitrogen atom carries an oxygen atom to form the corresponding N- oxide or when no nitrogen atom carries an oxygen atom, A is substituted by oxo on a ring carbon, or a pharmaceutically acceptable salt of such a compound.
• A is substituted by oxo on a ring carbon.
• A is pyrrolidino, piperidyl , piperidino, piperazinyl, pyridyl, pyrrolidino, pyrrolidinyl, morpholino, lower alkylpiperazino, N-methylpiperazino, 4-methyl-3-oxo-1 -piperazinyl, 3-oxo- 1 -piperazinyl, 1 H-imidazolyl, 1 H-2-methylimidazolyl, 1 H-4-methylimidazolyl or 1 H-2.4- dimethylimidazolyl, cyclohexyl or phenyl, optionally substituted by oxo on a ring carbon;
• A represents a piperazino group of the following formula A'
• R 3 represents, hydrogen, lower alkyl or acetyl.
• R is hydrogen
• R 2 is hydrogen, methyl or hydroxymethyl
• A is A', optionally substituted by oxo on a ring carbon
• R 3 is methyl or hydrogen, or salts of such compounds.
• A is preferably selected from lower alkyl- oxo-piperazino such as 4-methyl-3-oxo-1 -piperazinyl or oxo-piperazino such as 3-oxo-1 - piperazinyl, oxo-pyrrolidin, oxo-piperidino, oxo-piperidyl, oxo-morpholino, oxo-cyclohexyl, succinimido or glutarimido.
• lower alkyl- oxo-piperazino such as 4-methyl-3-oxo-1 -piperazinyl or oxo-piperazino such as 3-oxo-1 - piperazinyl, oxo-pyrrolidin, oxo-piperidino, oxo-piperidyl, oxo-morpholino, oxo-cyclohexyl, succinimido or glutarimido.
• the nitrogen atoms, which carry an oxygen atom to form the corresponding N-oxides are preferably the ring nitrogen atoms located on pyrimidine, pyrindinyl, "A” or piperazino group of the formula A'.
• R 5 R 6 By defining “R 5 R 6 together", the applicant does not include in the numbering the nitrogen, oxygen or carbon group mentioned in NR 5 R 6 , CR 5 R 6 or OR 5 R 6 .
• the prefix “lower” denotes a radical having up to and including a maximum of 7, especially up to and including a maximum of 4 carbon atoms, the radicals in question being either linear or branched with single or multiple branching.
• Lower alkyl is preferably alkyl with from and including 1 up to and including 7, preferably from and including 1 to and including 4, and is linear or branched; preferably, lower alkyl is butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl, ethyl or methyl.
• Preferably lower alkyl is methyl, propyl or tert-butyl.
• Hydroxy-lower alkyl preferably hydroxymethyl, 2-hydroxyethyl or 2-hydroxy-2-propyl.
• Lower alkoxy is especially methoxy, ethoxy, isopropyloxy, or tert-butyloxy.
• the invention relates to compounds of formula II
• R-i is hydrogen or hydroxy
• R 2 is lower alkyl or hydroxy-lower alkyl
• R 3 is hydrogen, methyl or acetyl
• the stars indicate the nitrogen atoms which optionally carry an oxygen atom to form the corresponding N-oxides, with the proviso that at least one of the three nitrogen atoms marked by a star carries an oxygen atom if R-* is hydrogen, R 2 is methyl and R 3 is hydrogen or methyl, or salts of such compounds.
• the nitrogen atoms of the 2-pyrimidine can also carry one or two oxygen atoms to form the corresponding N-oxides.
• a compound of formula II carries at least one oxygen atom to form the corresponding N-oxide.
• the piperazinyl is substituted by oxo to from a lower alkyl-oxo-piperazino such as 4-methyl-3-oxo-1 -piperazinyl or an oxo-piperazino such as 3-oxo-1 -piperazinyl.
• a lower alkyl-oxo-piperazino such as 4-methyl-3-oxo-1 -piperazinyl or an oxo-piperazino such as 3-oxo-1 -piperazinyl.
• R-) is hydroxy
• the 3-pyridinyl moiety is substituted by hydroxy at a ring carbon atom at position 2, 4, 5 or 6.
• Lower alkyl R 2 is preferably methyl.
• Hydroxy-lower alkyl R 2 is preferably hydroxymethyl.
• Salts are especially the pharmaceutically acceptable salts of compounds of formula I or II.
• Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids, from compounds of formula I or II with a basic nitrogen atom, especially the pharmaceutically acceptable salts.
• a compound of formula I or II possesses valuable pharmacological properties and may, for example, be used as an anti-tumour agent, as an agent to treat atherosclerosis, as an agent to treat restenosis, as an anti-leukemic agent for the prevention of transplantation-induced disorders, such as obliterative bronchiolitis, and/or for preventing the invasion of warmblooded animal cells by certain bacteria, such as Porphyromonas gingivalis.
• the phosphorylation of proteins has long been known as an essential step in the differentiation and division of cells. Phosphorylation is catalysed by protein kinases subdivided into serine/threonine and tyrosine kinases.
• the tyrosine kinases include PDGF (Platelet-derived Growth Factor) receptor tyrosine kinase.
• PDGF is a very commonly occurring growth factor, which plays an important role both in normal growth and also in pathological cell proliferation, such as is seen in carcinogenesis and in diseases of the smooth-muscle cells of blood vessels, for example in atherosclerosis and thrombosis.
• the inhibition of PDGF-stimulated receptor tyrosine kinase activity in vitro is measured in PDGF receptor immune complexes of A431 cells, as described by E. Andrejauskas- Buchdunger and U. Regenass in Cancer Research 52, 5353-5358 (1992).
• a compound of formula I or II inhibits PDGF-dependent acellular receptor phosphorylation.
• the inhibition of PDGF receptor tyrosine kinase is measured in a microtitre ELISA assay (cf Trinks et al., J. Med. Chem. 37, 1015-27 (1994).
• tumour diseases such as gliomas, sarcomas, prostate tumours, and tumours of the colon, breast, and ovary.
• a compound of formula I or II also inhibits cellular processes involving the so-called stem-cell factor (SCF, also known as the c-Kit ligand or steel factor), such as SCF receptor (Kit) autophosphorylation and the SCF-stimulated activation of MAPK kinase (mitogen-activated protein kinase).
• SCF stem-cell factor
• Kit SCF receptor
• a compound of formula I or II inhibits the tyrosine kinase activity of c-Kit.
• This can be shown in a tyrosine kinase inhibition assay using the cytoplasmatic kinase domain of c-Kit.
• the assay is performed as follows:
• the baculovirus donor vector pFbacGOI (GIBCO) is used to generate a recombinant baculovirus that expresses the amino acid region amino acids 544-976 of the cytoplasmic kinase domains of human c-Kit.
• the coding sequences for the cytoplasmic domain of c-Kit is amplified by PCR from a human uterus c-DNA library (Clontech).
• the amplified DNA fragment and the pFbacGOI vector are made compatible for ligation by digestion with BamH1 and EcoRI. Ligation of these DNA fragments results in the baculovirus donor plasmid c-Kit.
• the production of the viruses, the expression of proteins in Sf9 cells and the purification of the GST-fused proteins are performed as follows: Production of virus: Transfer vector (pFbacGOI -c-Kit) containing the c-Kit kinase domain is transfected into the DHIOBac cell line (GIBCO) and the transfected cells are plated on selective agar plates. Colonies without insertion of the fusion sequence into the viral genome (carried by the bacteria) are blue.
• Virus containing media is collected from the transfected cell culture and used for infection to increase its titre. Virus containing media obtained after two rounds of infection is used for large-scale protein expression. For large-scale protein expression 100 cm 2 round tissue culture plates are seeded with 5 x 10 7 cells/plate and infected with 1 mL of virus-containing media (approx. 5 MOIs). After 3 days the cells are scraped off the plate and centrifuged at 500 rpm for 5 min.
• Cell pellets from 10-20, 100 cm 2 plates, are resuspended in 50 mL of ice-cold lysis buffer (25 mM Tris-HCI, pH 7.5, 2 mM EDTA, 1% NP-40, 1 r ⁇ M DTT, 1 mM PMSF). The cells are stirred on ice for 15 min and then centrifuged at 5000 rpms for 20 min.
• ice-cold lysis buffer 25 mM Tris-HCI, pH 7.5, 2 mM EDTA, 1% NP-40, 1 r ⁇ M DTT, 1 mM PMSF.
• GST-tagged protein The centrifuged cell lysate is loaded onto a 2 mL glutathione-sepharose column (Pharmacia) and washed three times with 10 mL of 25 mM Tris-HCI, pH 7.5, 2 mM EDTA, 1 mM DTT, 200 mM NaCI.
• the GST-tagged protein is eluted by 10 applications (1 mL each) of 25 mM Tris-HCI, pH 7.5, 10 mM reduced-glutathione, 100 mM NaCI, 1 mM DTT, 10 % Glycerol and stored at -70°C.
• Tyrosine protein kinase assays with purified GST-c-Kit are carried out in a final volume of 30 ⁇ L containing 200-1800 ng of enzyme protein (depending on the specific activity), 20 mM Tris-HCI, pH 7.6, 3 mM MnCI 2 , 3 mM MgCI 2 , 1 mM DTT, 10 ⁇ M Na 3 VO 4 , 5 ⁇ g/mL poly(Glu,Tyr) 4:1 , 1 % DMSO, 1.0 ⁇ M ATP and 0.1 ⁇ Ci [ ⁇ 33 P] ATP.
• the activity is assayed in the presence or absence of inhibitors, by measuring the incorporation of 33 P from [ ⁇ 33 P] ATP into the poly(Glu,Tyr) 4:1 substrate.
• the assay (30 ⁇ L) is carried out in 96-well plates at ambient temperature for 20 min under conditions described below and terminated by the addition of 20 ⁇ L of 125 mM EDTA. Subsequently, 40 ⁇ L of the reaction mixture is transferred onto Immobilon-PVDF membrane (Millipore, Bedford, MA, USA) previously soaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5 % H 3 PO 4 and mounted on vacuum manifold with disconnected vacuum source.
• IC 50 values are calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at four concentrations (usually 0.01 , 0.1 , 1 and 10 ⁇ M).
• One unit of protein kinase activity is defined as 1 nmole of 33 P ATP transferred from [ ⁇ 33 P] ATP to the substrate protein per minute per mg of protein at 37 °C.
• a compound of formula I or II inhibits also the autophosphorylation of SCF receptor (and c- Kit, a proto-oncogen).
• Inhibition of the autophosphorylation of the SCF receptor can be measured using e.g. MO7e cells, a human promegakaryocytic leukaemia cell line which depends on SCF for proliferation. They are obtained from Grover Bagby, Oregon Health Sciences University, USA. The cells are cultivated in RPM1 1649 medium supplemented with 10 FBS and 2.5 ng/ml GC-CMF. GM-SCF and SCF are commercially available.
• Serum- deprived MO7e cells are prepared and incubated for 90 min at 37 °C with the test substance before being stimulated with recombinant SCF for 10 min at 37 °C.
• Identical quantities of cell lysates are analysed by Western blot using antiphosphotyrosine antibodies (Buchdunger et al., Proc. Natl. Acad. Sci (USA) 92, 2558-62 (1995)).
• the immunodecorated proteins are detected by means of the ECL Western blotting system from Amersham (Amersham, UK).
• a compound of formula I or II may be used not only as a tumour-inhibiting substance, for example in small cell lung cancer, but also as an agent to treat non-malignant proliferative disorders, such as atherosclerosis, thrombosis, psoriasis, scleroderma, and fibrosis, as well as for the protection of stem cells, for example to combat the haemotoxic effect of chemotherapeutic agents, such as 5-fluoruracil, and in asthma. It may especially be used for the treatment of diseases which respond to an inhibition of the PDGF receptor kinase.
• a compound of formula I or II prevents the development of multidrug resistance in cancer therapy with other chemotherapeutic agents or abolishes a pre-existing resistance to other chemotherapeutic agents.
• a compound of formula I or II may be used to advantage in combination with other antitumour agents, such as especially other c-Kit inhibitors and inhibitors of Vascular Endothelial Growth Factor (VEGF) receptor or c-Src activity.
• VEGF Vascular Endothelial Growth Factor
• Abl kinase is inhibited by a compound of formula I or II.
• the inhibition of v-Abl tyrosine kinase is determined by the methods of N. Lydon et al., Oncogene Research 5, 161-173 (1990) and J. F. Geissler et al., Cancer Research 52, 4492-8 (1992). In those methods [Val 5 ]-angiotensin II and [ ⁇ 32 P]-ATP are used as substrates.
• a compound of formula I or II also inhibits Bcr-Abl kinase (see Nature Medicine 2, 561-566 (1996)) and is thus suitable for the treatment of Bcr-Abl-positive cancer and tumour diseases, such as leukaemias (especially chronic myeloid leukaemia and acute lymphoblastic leukaemia, where especially apoptotic mechanisms of action are found), and also shows effects on the subgroup of leukaemic stem cells as well as potential for the purification of these cells in vitro after removal of said cells (for example, bone marrow removal) and reimplantation of the cells once they have been cleared of cancer cells (for example, reimplantation of purified bone marrow cells).
• Bcr-Abl kinase see Nature Medicine 2, 561-566 (1996)
• Bcr-Abl-positive cancer and tumour diseases such as leukaemias (especially chronic myeloid leukaemia and acute lymphoblastic leukaemia, where especially apoptotic mechanisms of action are found)
• Test for activity against c-Abl protein tyrosine kinase The test is conducted as a filter binding assay as follows: The His-tagged kinase domain of c-Abl is cloned and expressed in the baculovirus/Sf9 system as described by Bhat et al., J. Biol. Chem. 272, 16170-5 (1997). A protein of 37 kD (c-Abl kinase) is purified by a two-step procedure over a cobalt metal chelate column followed by an anion exchange column with a yield of 1 -2 mg/L of Sf9 cells.
• the purity of the c-Abl kinase is >90% as judged by SDS-PAGE after Coomassie blue staining.
• the assay contains: c-Abl kinase (50 ng), 20 mM Tris-HCI, pH 7.5, 10 mM MgCI 2 , 10 ⁇ M Na 3 VO 4 , 1 mM DTT and 0.06 vCi/assay [ ⁇ 33 P]-ATP (5 ⁇ M ATP) using 30 g/mL poly- Ala,Glu,Lys,Tyr-6:2:5:1 (Poly-AEKY, Sigma P1152) in the presence of 1% DMSO, total volume of 30 ⁇ L.
• Reactions are terminated by adding 10 ⁇ L of 250 mM EDTA, and 30 ⁇ L of the reaction mixture is transferred onto Immobilon-PVDF membrane (Millipore, Bedford, MA, USA) previously soaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5% H 3 PO 4 and mounted on vacuum manifold with disconnected vacuum source. After spotting all samples, vacuum is connected and each well rinsed with 200 ⁇ L 0.5 % H 3 PO 4 . Membranes are removed and washed on a shaker with 0.5% H 3 PO 4 (4 times) and once with ethanol. Membranes are counted after drying at ambient temperature, mounting in Packard TopCount 96-well frame, and addition of 10 /L/well of Microscint TM (Packard).
• the murine myeloid progenitor cell line 32Dcl3 transfected with the p210 Bcr-Abl expression vector pGDp210Bcr/Abl (32D-bcr/abl) was obtained from J. Griffin (Dana Faber Cancer Institute, Boston, MA, USA).
• the cells express the fusion Bcr-Abl protein with a constitutively active Abl kinase and proliferate growth factor independent.
• the cells are expanded in RPM1 1640 (AMIMED), 10% fetal calf serum, 2 mM glutamine (Gibco) (..complete medium”), and a working stock is prepared by freezing aliquots of 2 x 10 6 cells per vial in freezing medium (95% FCS, 5% DMSO (SIGMA)). After thawing, the cells are used during maximally 10 -12 passages for the experiments.
• the compound is incubated together with the cells for 90 min at 37°C, 5% CO 2 , followed by centrifugation of the tissue culture plates at 1300 rpm (Beckman GPR centrifuge) and removal of the supernatants by careful aspiration taking care not to remove any of the pelleted cells.
• the cell pellets are lysed by addition of 150 ⁇ L lysis buffer (50 mM Tris/HCI, pH 7.4, 150 mM sodium chloride, 5 mM EDTA, 1 mM EGTA, 1% NP-40, 2 mM sodium ortho- vanadate, 1 mM PMSF, 50 g/mL aprotinin and 80 ⁇ g/mL leupeptin) and either used immediately for the ELISA or stored frozen in the plates at -20°C until usage.
• Black ELISA plates (Packard HTRF-96 black plates) are precoated over night at 4°C with 50 ng/well of the rabbit polyclonal anti-abl-SH3 domain Ab 06-466 from Upstate in 50 ⁇ L PBS.
• the difference between the ELISA-readout (CPS) obtained for with the lysates of the untreated 32D-Bcr/Abl cells and the readout for the assay-background (all components, but without cell lysate) is calculated and taken as 100% reflecting the constitutively phosphorylated Bcr-Abl protein present in these cells.
• the activity of the compound on the Bcr-Abl kinase activity is expressed as percent reduction of the Bcr-Abl phosphorylation.
• the values for the IC 50 and IC 90 are determined from the dose response curves by graphical extrapolation.
• Test for activity against mutant Bcr-Abl The activity of compounds on the M351T mutant Bcr-Abl kinase activity is assessed as described above, except that 32Dcl3 cells transfected with mutant Bcr-Abl in place of p210 Bcr-Abl are utilised.
• c-Raf-1 protein kinase assay Recombinant c-Raf-1 protein is obtained by triple infection of Sf21 cells with GST-c-Raf-1 recombinant baculovirus together with v-Src and v-Ras recombinant baculoviruses that are required for active c-Raf-1 kinase production (Williams et al., PNAS 1992; 89:2922-6). Active Ras (v-Ras) is required to recruit c-Raf-1 to the cell membrane and v-Src to phosphorylate c-Raf-1 to fully activate it.
• Cells are seeded at 2.5 x 10 7 cells per 150 mm dish and allowed to attach to a 150 mm dish for 1 hr at RT.
• Media SF900II containing 10 % FBS
• baculovirus GST-c-Raf-1 , v- Ras and v-Src are added at MOI of 3.0, 2.5 and 2.5, respectively, in a total volume of 4- 5 mL.
• Cells are incubated for 1 hr at RT and then 15 mL of medium is added. Infected cells are incubated for 48-72 hr at 27°C.
• Infected Sf21 cells are scraped and collected into a 50 mL tube and centrifuged for 10 min at 4°C at 1100 g in a Sorvall centrifuge.
• the cell pellet is washed once with ice cold PBS and lysed with 0.6 mL lysis buffer per 2.5 x 10 7 cells. Complete lysis of cells is achieved after 10 min on ice with occasional pipetting.
• the cell lysates are centrifuged for 10 min at 4°C at 14,500 g in a Sorvall centrifuge with SS-34 rotor and the supernatant is transferred to a fresh tube and stored at -80°C.
• c-Raf-1 is purified from cell lysates using 100 ⁇ L of packed glutathione-sepharose 4B beads equilibrated in ice cold PBS per 2.5 x 10 7 cells.
• GST-c-Raf-1 is allowed to bind to the beads at 4°C for 1 hr with rocking. Bound GST-c-Raf-1 with beads is transferred to a column. The column is washed once with lysis buffer and twice with ice cold Tris buffered saline. Ice cold elution buffer is added and column flow is stopped to allow the free glutathione to disrupt the interaction of GST-c-Raf-1 with glutathione sepharose beads. Fractions (1 mL) are collected into pre- chilled tubes. Each tube contains 10 % glycerol (final concentration) to maintain kinase activity during freeze thaw cycles. Purified fractions of GST-c-Raf-1 kinase protein are stored at -80°C.
• IKB is used as substrate for the c-Raf-1 kinase.
• l ⁇ B is expressed in bacteria as a His-tagged protein BL21.
• LysS bacteria containing the IKB plasmid are grown to an OD600 of 0.6 in LB medium, then induced to express the IKB with IPTG (final concentration of 1 mM) for 3 hrs at 37°C and then bacteria are lysed by sonication (microtip limit setting for 3 times at 1 min each in sonication buffer [50 mM Tris pH 8.0, 1 mM DTT, 1 mM EDTA] and centrifuged at 10,000 g for 15 min. The supernatant is mixed with ammonium sulfate to give a final concentration of 30 %.
• the assay is carried out in 96- well plates at ambient temperature for 60 min. It contains (total volume of 30 ⁇ L): c-Raf-1 kinase (400 ng), 25 mM Tris-HCI, pH 7.5, 5 mM MgCI 2 , 5 mM MnCI 2 , 10 ⁇ M Na 3 VO 4 , 1 mM DTT and 0.3 ⁇ Ci/assay [ ⁇ 33 P]-ATP (10 ⁇ M ATP) using 600 ng IKB in the presence of 1 % DMSO.
• Reactions are terminated by adding 10 ⁇ L of 250 mM EDTA and 30 ⁇ L of the reaction mixture is transferred onto Immobilon-PVDF membrane (Millipore, Bedford, MA, USA) previously soaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5 % H 3 PO 4 and mounted on vacuum manifold with disconnected vacuum source. After spotting all samples, vacuum is connected and each well rinsed with 200 ⁇ L 0.5 % H 3 PO 4 . Membranes are removed and washed 4 x on a shaker with 0.5 % H 3 PO 4 , once with ethanol. Membranes are counted after drying at ambient temperature, mounting in Packard TopCount 96-well frame, and addition of 10 ⁇ L/well of Microscint TM (Packard).
• hypoxia-selective products due to bio-reduction (deoxygenation) in the cell especially in the tumours and in the brain.
• Hypoxia-activated pro- drugs are especially useful in cancer therapy since severe hypoxia occurs in solid tumour tissue or in the brain. Hypoxic cells can be exploited for therapy by non-toxic, hypoxia- activated pro-drugs.
• Caco-2 cell monolayers grown on polyethylene terephthalate (PET) filters (FalconTM) for 21- 25 days are used for transport experiments.
• the flux of compounds across Caco-2 cell monolayers grown on PET filters as well as across PET filters alone without Caco-2 cells (for system validation) in the presence and absence of the potent efflux pump inhibitors CsA and Verapamil, respectively, are determined as follows: Prior to the transport experiment, the culture medium in the acceptor compartment (0.2 ml for apical and 1.0 ml for basolateral sides) is replaced with acceptor solution (HBSS, when relevant containing the inhibitor of interest) preincubated at 37 9 C.
• acceptor solution HBSS
• the medium in the donor compartment (0.35 ml for apical and 1.15 ml for basolateral sides) is replaced with donor solution (compound in HBSS, when relevant containing inhibitor of interest) preincubated at 37°C. Aliquots of 150 l are removed from the donor and the acceptor side after about 1 and 120 minutes. Transport experiments in both apical-to-basolateral and basolateral-to-apical directions are performed in triplicate at 37 9 C in an incubator without shaking.
• the plasma protein binding of the compounds of formula I or II is superior with regard to free fraction and/or association with plasma proteins (e.g. albumin, - 1 -acid glycoprotein (AAG)), to that observed with compound A.
• a lower extent of association to AAG results in less pronounced variability of free fraction of N-oxides and also has an effect of free fraction of compound A compounds.
• the free fraction of compound A ranges from 4 to 5%.
• compound A was mainly found associated with albumin and alpha-1-acid glycoprotein (AAG).
• the fraction associated with lipoproteins and gamma globulins was ⁇ 5%.
• the reduced plasma protein-binding of compounds of formula I or II is shown by the following example.
• the free (or unbound) fraction of compounds of formula I or II is determined by the ultracentrifugation method which was used also for compound A (see European patent application No. 1250140 or International patent application WO 01/47507 filed on 22/12/2000).
• Solutions of human serum albumin (40 g/L) and ⁇ -1 -acid glycoprotein (1 g/L) will be prepared in Soerensen buffer pH 7.4 containing 0.9% NaCI (w/v).
• 30 ⁇ L of the compounds of formula I or II stock solutions are directly spiked into 3 mL of the protein solutions to get the intended final concentrations of 300-5000 ng/mL compounds of formula I or II (ethanol final concentration 0.5%, factor 1 :200).
• the pharmacokinetics of the compounds of formula I or II are advantageous over compound A with regard to C ma ⁇ (highest observed concentration in plasma in units mass/volume), half-live (refers to the time after administration of the drug to observe a diminution of one half of the measured pharmacological response; In one aspect, the half-life is enhanced when the half-life is increased by at least 50%) or AUC (plasma concentrations over time, as defined by the Area Under the Curve (AUC) in units of mass-time/volume) in plasma as a matter of transport mechanism (e.g. P-gp).
• AUC Area Under the Curve
• P-gp Area Under the Curve
• This advantageous pharmacokinetics is shown by giving to animals (e.g.
• the reduced plasma protein binding of the compounds of formula I or II to plasma proteins can cause an increase in the apparent volume of distribution due to higher fraction of unbound drug (fu).
• the distribution of the compounds of formula I or II into organs and tissues (including the brain) is different from that of compound A. This can be shown as follows.
• Animals receive a single dose of compounds of formula I or II (one group of animals is treated intravenously and one group of animals is treated per orally). Animals are sacrificed at selected time points (e.g. 0.083 min (iv) and 1 , 8, 24 and 48 h after iv or po dosing). Brain of treated animals are taken, homogenates of brain are prepared and samples are prepared (e.g. extraction of homogenate with organic solvent such as methanol, acetonitrile or others) for analysis (HPLC/UV or LC-MS) of compounds of formula I or II and compound A. Concentrations of compounds of formula I or II and compound A in brain and plasma is measured for determination of ratio brain/plasma.
• organic solvent such as methanol, acetonitrile or others
• radiolabelled N-oxide e.g. [ 14 C]-label; 100 ⁇ Ci/kg b.w.
• the uptake / distribution of radioactive substance(s) throughout the body of the animal is investigated using quantitative whole-body autoradioluminography (QWABL).
• QWABL quantitative whole-body autoradioluminography
• the animals arer sacrificed at selected time points and frozen in a mixture of dry-ice and hexane at approx. -75°C.
• Frozen animals are embedded in a pre-cooled 2% aqueous gel of Na-CMC, at approx. -75°C; . 40 ⁇ m thick sections are obtained at ca.
• Scan is performed in a Fuji BAS 2000 TR phosphor imager, immediately after the end of the exposure, under controlled light conditions, at a 100 ⁇ m scanning step with a 1024 gradation.
• Image analysis is done as follows: The resulting photostimulated light data files are corrected by subtracting the background, processed electronically with the help of a MCID/M4 (3.0 Rev. 1.3) image analyzer (Imaging Research, St. Catherines, Ontario, Canada) and automatically converted into radioactivity concentrations using a 1st degree polynomial calibration curve obtained from a radioactive blood scale processed under similar conditions as the samples.
• the size of the measurement areas are the same as that of each blood standard of the blood scale used to set the calibration curve. Image files processed using the Adobe Photoshop® software. .
• the potential inhibitor is added with increasing concentrations and the metabolic reactions are evaluated by the corresponding analytical method (LC/MS, HPLC, Fluorescence).
• the conversion rate without inhibitor is set to 100 % and the inhibition rate is evaluated as the concentration of inhibitor needed to suppress 50 % of the conversion (IC50).
• the following marker substrates are used:
• Acetanilide (mostly applied in hepatocytes)
• Testosterone strongly Felodipine recommended to use at Cyclosporin least two structurally Terfenadine unrelated substrates
• a compound of formula I or II shows useful effects in the treatment of disorders arising as a result of transplantation, for example, allogenic transplantation, especially tissue rejection, such as especially obliterative bronchiolitis (OB), i.e. a chronic rejection of allogenic lung transplants.
• allogenic transplantation especially tissue rejection, such as especially obliterative bronchiolitis (OB), i.e. a chronic rejection of allogenic lung transplants.
• OB obliterative bronchiolitis
• those with OB often show an elevated PDGF concentration in bronchoalveolar lavage fluids.
• a compound of formula I or II is administered to rats with tracheal allogenic transplants, for example in a dose of 50 mg/kg i.p., it can be shown after removal of 10 transplants per group after 10 and 30 days for morphometric analysis of possible epithelial lesions and occlusion of the airways, and investigation for immunohistochemical pathways of action that, although a compound of formula I or II has no significant effect on epithelial necrosis or infiltration by inflammatory cells, it does markedly reduce fibroproliferation and occlusion of the lumen compared with controls.
• CsA cyclosporin A
• rapamycin or ascomycin
• immunosuppressant analogues thereof for example cyclosporin G, FK-506 or comparable compounds
• corticosteroids cyclophosphamide
• azathioprine methotrexate
• brequinar leflunomide
• mizoribine mycophenolic acid
• mycophenolate mofetil 15- deoxyspergualin
• immunsuppressant antibodies especially monoclonal antibodies for leucocyte receptors, for example MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands
• other immunomodulatory compounds such as CTLA4lg.
• a compound of formula I or II is also effective in diseases associated with vascular smooth- muscle cell migration and proliferation (where PDGF and PDGF receptor often also play a role), such as restenosis and atherosclerosis.
• diseases associated with vascular smooth- muscle cell migration and proliferation where PDGF and PDGF receptor often also play a role
• diseases associated with vascular smooth- muscle cell migration and proliferation such as restenosis and atherosclerosis.
• vascular smooth-muscle cells in vitro and in vivo can be demonstrated by administration of a compound of formula I or II and also by investigating its effect on the thickening of the vascular intima following mechanical injury in vivo.
• a compound of formula I or II is used in 0.1 N HCI or DMSO at a concentration of 10 mM for in vitro studies. The stock solution is further diluted with cell culture medium and used in concentrations of 10 to 0.1 ⁇ M for the experiments.
• a compound of formula I or II is dissolved for example in DMSO at a concentration of 200 mg/ml and then diluted 1 :20 with 1% Tween in 0.9% saline solution. After sonication, a clear solution is obtained. The stock solutions are prepared fresh each day before administration. (The compound of formula I or II may also be dissolved simply in deionised water for oral administration or in 0.9% saline solution for parenteral administration). Administration is carried out 24 hours before the operation. A compound of formula I or II is administered to rats in one dose of 50 mg/kg i.p. per day for the entire observation period. Control rats are given the same formulation but without the presence of a compound of formula I or II. Oral administration is also possible.
• the primary cells are used for the experiments after passages 2 to 6. Subcultures are kept in DMEM (Dulbecco's Modified Eagle's Medium), supplemented with 10% fetal calf serum, 2 mmol/ml glutamine, 100 mmol/ml streptomycin, and 100 lU/ml penicillin. For identification purposes, the cells are left to grow on glass slide covers and stained immunohistochemically using an anti- -actin antibody obtained from smooth-muscle cells (see below).
• the migration of smooth-muscle cells is quantified in vitro using a Transwell cell culture insert (Costar, Cambridge, MA) whose upper and lower compartments are separated by a polycarbonate membrane of 8 ⁇ m pore size.
• the cells (100 ⁇ l at a concentration of 1 million cells/ml) are exposed in the upper compartment.
• 60 ng/ml PDGF-BB or PDGF-AA (Upstate Biotechnology Inc., Lake Placid, NY) is added to the lower compartment, supplemented with 0.5% fetal calf serum and 0.1% bovine serum albumin, and the test compound of formula I or II is added in concentrations of 3, 1 , 0.3, 0.1 , 0.03, 0.01 , and 0.003 ⁇ M.
• the Transwell chambers are covered with f ibronectin at a concentration of 10 ⁇ g/ml for 24 h at 4 °C (human cellular fibronectin, Upstate Biotechnology Inc.).
• the filters are removed, fixed in methanol, and stained with Mayer's haematoxylin and eosin.
• the migrated cells on the lower side of the filter membrane are determined by counting the specified sectional fields on the filters with the aid of a light microscope with a magnification of 400x.
• the inhibition of migration is quantified in terms of the percentage of cells versus with the control.
• the viability of the cells is tested by incorporation of 3H-thymidine in DMEM, supplemented with 10% fetal calf serum.
• An inhibition of migration induced by PDGF-AA and especially by PDGF-BB is observed with a compound of formula I or II.
• mice the aorta and carotid artery of male Wistar rats (purchased from the Laboratory Animal Centre of the University of Helsinki, Finland) are denuded.
• the rats are anaesthetised with 240 mg/kg chloral hydrate i.p. and Buprenorphine (Temgesic, Reckitt & Coleman, Hull, UK) is administered for perioperative and postoperative alleviation of pain. All animals are given human care in keeping with the "Principles of Laboratory Animal Care” and the "Guide for the Care and Use of Laboratory Animals" of the NIH (NIH Publication 86- 23, revised 1985). Rats weighing 200-300 g were used for the denudation procedure.
• the left common carotid artery is denuded of endothelium through the intraluminal passage of a 2F embolectomy catheter (Baxter Healthcare Corporation, Santa Ana, CA, 27).
• a 2F embolectomy catheter Baxter Healthcare Corporation, Santa Ana, CA, 27.
• the catheter is passed through the lumen three times, inflated with 0.2 ml air.
• the external carotid is ligated after removal of the catheter and the wound closed.
• the histological changes are evaluated by reference to sections of mid-carotid 4 days after denudation.
• the thoracic aorta is denuded of endothelium using a 2F Fogarty arterial embolectomy catheter.
• the catheter is inserted into the thoracic aorta via the left iliac artery, inflated with 0.2 ml air, and passed through the lumen five times to remove the endothelium.
• the iliac artery is then ligated. Three times (3, 7 and 14 days) are selected for evaluation of the histological changes.
• the rats were given 3 x 0.1 ml BrdU-labelling reagent at 8-hour intervals over a period of 72-96 hours after the operation.
• a third group of rats is given a pulsed dose of 0.3 ml BrdU three hours before sacrifice.
• Histological samples are fixed in 3% paraformaldehyde solution for 4 h for embedding in paraffin. Morphological changes are evaluated from paraffin sections stained with Mayer's haematoxylin-eosin. The cell counts of different vessel sections are calculated at a magnification of 400x.
• immunohistochemical staining of acetone-fixed samples is carried out using an anti- ⁇ -actin antibody obtained from smooth-muscle cells (Bio-Makor, Rehovot, Israel). Primary smooth-muscle cells are identified on acetone-fixed glass cover slides using the same staining method.
• the sections are incubated with the primary antibody (dilution 1 :2000), washed, and incubated consecutively with peroxidase-conjugated rabbit- antimouse-lg and goat-antirabbit-lg, followed by treatment with substrate solution with the chromogen 3-amino-9-ethylcarbazol and hydrogen peroxide.
• BrdU stains are prepared from paraffin sections using a primary mouse antibody (Bu20a, Dako, A/S, Denmark) and the Vectastain Elite ABC-Kit (Vector Laboratories, Burliname, CA).
• the sections are deparaffinised and treated by microwave at 500 W (2 x 5 min in 0.1 M citrate buffer, pH 6), followed by treatment with 95% formamide in 0.15 M trisodium citrate for 45 min at 70 °C.
• Antibody dilutions are prepared according to the manufacturer's specifications.
• the sections are counterstained with Mayer's haematoxylin and eosin, and positive cells are counted separately for the initima, media, and adventitia.
• a compound of formula I or II is also capable of inhibiting angiogenesis. This may be demonstrated as follows: a chamber containing agar (0.8%) and heparin (2 U/ml) with or without growth factor (VEGF 3 ⁇ g/ml, PDGF 1 ⁇ g/ml or bFGF 0.3 ⁇ g/ml) is implanted subcutaneously into normal mice (C57 BL/6). A compound of formula I or II is administered orally in a dose showing good anti-tumour activity in a nude mouse xenotransplant model. Dosing is started one day before implantation of the chambers. The chambers are removed after 5 days.
• the angiogenic efficacy is quantified by measuring both the vascularised tissue which has grown around the implant and the blood content of this tissue (external blood).
• the blood is determined by measuring the haemoglobin. Although the vessels do not grow into the agar, the agar becomes intensely red if an antiangiogenic effect is present. If a compound inhibits the increase in blood that is induced by the growth factor, this is seen as an indication that the compound in question is blocking the angiogenic effect of the growth factor concerned. Inhibition of the weight but not the volume of blood suggests an effect on the proliferation of fibroblasts. A suppression of the control response suggests an inhibition of wound healing.
• a compound of formula I or II inhibits the angiogenic effect of all three growth factors (VEGF, PDFG, bFGF).
• R-i is hydrogen
• R 2 is methyl or hydroxymethyl
• R 3 is methyl
• the stars indicate the nitrogen atoms which optionally carry an oxygen atom to form the corresponding N-oxides, with the proviso that at least one of the three nitrogen atoms marked by a star carries an oxygen atom if R 2 is methyl, or salts of such compounds.
• R-i is hydrogen
• R 2 is hydroxy-lower alkyl
• R 3 is methyl, and the stars indicate the nitrogen atoms which optionally carry an oxygen atom to form the corresponding N-oxides, or salts of such compounds.
• A has the meanings given under formula I. and a compound thus obtained is converted into a N-oxide of formula I with a suitable oxidizing agent or if not converted into a N-oxide, A has to be substituted by oxo on a ring carbon;
• R-i and R_ have the meanings given under formula II and the star indicates a nitrogen atom which optionally carries an oxygen atom, is reacted with a compound of formula IV
• R 3 has the meanings given under formula II and the stars indicate the nitrogen atoms which optionally carry an oxygen atom;
• R-- and R 2 have the meanings given under formula I, Hal is halo (e.g. -Cl, -Br, -F, -I), is reacted with a compound of formula VI
• R ⁇ and R 2 have the meanings given under formula II, Hal is halo (e.g. -Cl, -Br, -F, -I) and the star indicates a nitrogen atom which optionally carries an oxygen atom, is reacted with a compound of formula VI
• R 3 has the meanings given under formula II and the stars indicate the nitrogen atoms which optionally carry an oxygen atom;
• a compound thus obtained is optionally converted into a N-oxide of formula II with a suitable oxidizing agent; whereby functional groups which are present in the starting compounds of process a) or b) and are not intended to take part in the reaction, are present in protected form if necessary, and protecting groups that are present are cleaved, whereby the said starting compounds may also exist in the form of salts provided that a salt-forming group is present and a reaction in salt form is possible;
• a compound of formula I or II obtained by process a) or b) is converted into another compound of formula I or II, an obtained free compound of formula I or II is converted into a salt, an obtained salt of a compound of formula I or II is converted into the free compound or another salt, and/or a mixture of isomeric compounds of formula I or II is separated into the individual isomers.
• compounds of formula I or II are in a substantially pure form.
• substantially pure is understood in the context of the present invention to mean substantially free of biological material such as found in the blood, especially less than 10%, preferably less than 1 %, and most preferably free of such biological material.
• a suitable oxidizing agent for converting a compound obtained by process a) or b) into a N- oxide of formula I or II is preferably hydrogen peroxide or a suitable peracid, for example a suitable perbenzoic acid, such as especially m-chloro-perbenzoic acid.
• the reaction is carried out in an inert solvent, for example a halogenated hydrocarbon, such as dichloromethane, at temperatures of approximately from -20 °C to the boiling point of the solvent in question, in general below +100 °C. If hydrogen peroxide is used as the oxidizing agent, the reaction is preferably carried out in water at about room temperature.
• the desired N-oxide can then be purified using conventional methods such as e.g. column chromatography or recrystallisation.
• N-oxides of formula I or II may be prepared according to the process described in the preceding paragraph by already oxidizing the starting materials used in the synthesis of compounds of formula I or II.
• the reaction between a compound of formula III and a compound of formula IV preferably takes place in a suitable inert solvent, especially ⁇ /, ⁇ /-dimethylformamide, in the presence of propylphosphonic anhydride (Fluka, Buchs, Switzerland) and a base such as especially triethylamine, preferably at room temperature.
• a suitable inert solvent especially ⁇ /, ⁇ /-dimethylformamide
• propylphosphonic anhydride Fluka, Buchs, Switzerland
• a base such as especially triethylamine
• the reaction between a compound of formula V and a compound of formula IV preferably takes place in a suitable inert solvent, especially alcohols, e.g. lower alcohols such as especially ethanol, at elevated temperature, preferably near the boiling temperature of the solvent employed.
• a suitable inert solvent especially alcohols, e.g. lower alcohols such as especially ethanol
• Halo present in a compound of formula V is e.g. fluoro, chloro, bromo and iodo, preferably chloro.
• protecting groups of the starting compounds which should not take part in the reaction may be present in unprotected form or may be protected for example by one or more protecting groups.
• the protecting groups are then wholly or partly removed according to one of the known methods. Protecting groups, and the manner in which they are introduced and removed are described, for example, in "Protective Groups in Organic Chemistry", Plenum Press, London, New York 1973, and in “Methoden der organischen Chemie", Houben-Weyl, 4th edition, Vol. 15/1, Georg-Thieme-Verlag, Stuttgart 1974 and in Theodora W. Greene, "Protective Groups in Organic Synthesis", John Wiley & Sons, New York 1981.
• a characteristic of protecting groups is that they can be removed readily, i.e. without the occurrence of undesired secondary reactions, for example by solvolysis, reduction, photolysis or alternatively under physiological conditions.
• All process steps described here can be carried out under known reaction conditions, preferably under those specifically mentioned, in the absence of or usually in the presence of solvents or diluents, preferably those that are inert to the reagents used and able to dissolve them, in the absence or presence of catalysts, condensing agents or neutralising agents, for example ion exchangers, typically cation exchangers, for example in the protonated (H+-) form, depending on the type of reaction and/or reactants at reduced, normal, or elevated temperature, for example in the range from -100 °C to about 190 °C, preferably from about -80 °C to about 150 °C, for example at -80 to -60 °C, at RT, at -20 to 40 °C, at 0 to 100 °C or at the boiling point of the solvent used, under atmospheric pressure or in a closed vessel, if need be under pressure, and/or in an inert, for example an argon or nitrogen, atmosphere.
• the invention relates also to those embodiments of the process in which one starts from a compound obtainable at any stage as an intermediate and carries out the missing steps, or breaks off the process at any stage, or forms a starting material under the reaction conditions, or uses said starting material in the form of a reactive derivative or salt, or produces a compound obtainable by means of the process according to the invention under those process conditions, and further processes the said compound in situ.
• a compound of formula I or II is prepared according to the processes and process steps defined in the Examples.
• the compounds of formula I or II, including their salts, are also obtainable in the form of hydrates, or their crystals can include for example the solvent used for crystallisation (present as solvates).
• New starting materials and/or intermediates, as well as processes for the preparation thereof, are likewise the subject of this invention.
• such starting materials are used and reaction conditions so selected as to enable the preferred compounds to be obtained.
• the starting materials used in the above described process are known, capable of being prepared according to known processes (see also EP 0 564 409 B1), or commercially obtainable; in particular, they can be prepared using processes as described in the Examples.
• a compound of formula III wherein R 2 is lower alkyl and the nitrogen atom marked by a star does not carry an oxygen atom as a substituent can be prepared as described in EP 0 564 409 B1. Such compounds may then be converted into the corresponding N-oxides using a suitable oxidizing agent as described above under "Description of the process variants".
• a compound of formula III wherein R 2 is hydroxy-lower alkyl can be prepared analogously to Example 1 by starting with a compound of the following formula VII:
• the remaining starting materials are known, capable of being prepared according to known processes like those described in e.g. EP 0 564 409 B1 , or commercially available; or in particular, they can be prepared using processes as described in the Examples.
• Suitable N- phenyl -2-pyrimidine-amine derivatives to form the corresponding N-oxides are also described in e.g. EP 0 564 409 B1.
• the invention relates also to a process for the treatment of warm-blooded animals, including humans, suffering from said diseases, especially a tumour disease, wherein a quantity of a compound of formula I or II which is effective against the disease concerned, especially a quantity with antiproliferative and especially tumour-inhibiting efficacy, is administered to warm-blooded animals, including humans, in need of such treatment.
• the invention relates moreover to the use of a compound of formula I or II for the inhibition of the above- mentioned tyrosine kinases, especially PDGF receptor kinase, v-Abl kinase, and/or c-Kit receptor kinase, or for the preparation of pharmaceutical compositions for use in treating warm-blooded animals, including humans, especially for the treatment of tumours, such as gliomas, ovarian tumours, prostate tumours, colon tumours, and tumours of the lung, such as especially small cell lung carcinoma, and tumours of the breast or other gynaecological tumours.
• tumours such as gliomas, ovarian tumours, prostate tumours, colon tumours, and tumours of the lung, such as especially small cell lung carcinoma, and tumours of the breast or other gynaecological tumours.
• effective doses for example daily doses of about 1-2500 mg, preferably 1-1000 mg, especially 5-500 mg, are administered to warm-blooded animals, including humans, of about 70 kg bodyweight.
• the present invention relates to the use of N-phenyl -2-pyrimidine-- amine derivatives in which at least one nitrogen atom carries an oxygen atom to form the corresponding N-oxides or a pharmaceutically acceptable salt of such a compound for the preparation of a pharmaceutical composition for the treatment of a proliferative disorder.
• the present invention relates to the use of a compound of formula I or II or a pharmaceutically acceptable salt of such a compound for the preparation of a pharmaceutical composition for the treatment of a proliferative disorder.
• the proliferative disorder is selected from tumors or brain proliferative disorders.
• the invention further provides a method of treating warm-blooded animals, including humans, which comprises administering to such a warm-blooded animal suffering from a proliferative disorder, in a dose effective against said disorder, a compound of formula I or II or a pharmaceutically acceptable salt of such a compound.
• the instant invention provides a pharmaceutical composition comprising at least one N-phenyl -2-pyrimidine-amine derivative in which at least one nitrogen atom carries an oxygen atom to form the corresponding N-oxides or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
• the instant invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula I or II or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
• compositions of the present invention may contain at least one additional pharmaceutically active compound such as 4-(4-methylpiperazin-1 -ylmethyl)-N-[4-methyl-3- (4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide.
• additional pharmaceutically active compound such as 4-(4-methylpiperazin-1 -ylmethyl)-N-[4-methyl-3- (4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide.
• a pharmaceutical composition for the treatment of a proliferative disorder in warm-blooded animals, including humans comprising as an active ingredient a compound of formula I or II according or a pharmaceutically acceptable salt of such a compound, together with a pharmaceutically acceptable carrier.
• the invention relates also to pharmaceutical compositions comprising as an active ingredient a compound of formula I or II together with a pharmaceutically acceptable carrier, especially for the prevention or treatment of one of the said diseases, said pharmaceutical compositions being suitable for e.g. topical, enteral, for example oral or rectal, or parenteral administration.
• a pharmaceutically acceptable carrier especially for the prevention or treatment of one of the said diseases
• said pharmaceutical compositions being suitable for e.g. topical, enteral, for example oral or rectal, or parenteral administration.
• diluents for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycerin
• lubricants for example silica, talc, stearic acid, or salts thereof, typically magnesium or calcium stearate, and/or polyethylene glycol, are used for oral administration.
• Tablets may likewise contain binders, for example magnesium aluminium silicate, starches, typically corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and, if so desired, disintegrants, for example starches, agar, alginic acid, or a salt thereof, typically sodium alginate, and/or effervescent mixtures, or adsorbents, colouring agents, flavours, and sweetening agents.
• binders for example magnesium aluminium silicate, starches, typically corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and, if so desired, disintegrants, for example starches, agar, alginic acid, or a salt thereof, typically sodium alginate, and/or effervescent mixtures, or adsorbents, colouring agents, flavours, and sweetening agents.
• Such solutions are preferably isotonic aqueous solutions or suspensions, these possibly being prepared before use, for example in the case of lyophilised preparations containing the active substance either alone or together with a carrier, for example mannitol.
• the pharmaceutical substances may be sterilised and/or may contain excipients, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for the regulation of osmotic pressure, and/or buffers.
• compositions which, if so desired, may contain further pharmacologically active substances, such as other c-Kit inhibitors or inhibitors of VEGF receptor or c-Src activity, are prepared in a manner known ⁇ er se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilising processes, and contain from about 1 % to 100%, especially from about 1 % to about 20%, of the active substance or substances.
• pharmacologically active substances such as other c-Kit inhibitors or inhibitors of VEGF receptor or c-Src activity
• Example 1 4-IT4-Methyl-1 -piperazinyl)-methvn- ⁇ /-f4-hvdroxymethyl-3-rr4-(3-pyridinyl)-2- pyrimidinv ⁇ -aminol-phenvD-benzamide
• the cooled mixture is then concentrated to a volume of circa 150 mL by rotary evaporation under reduced pressure to give a suspension.
• the suspension is filtered and the precipitate is redissolved in ethyl acetate (500 mL) and washed with saturated aqueous sodium hydrogen carbonate (2 x 150 mL).
• the solution is dried (Na 2 SO 4 ), filtered and the solvent is evaporated off under reduced pressure to yield the crude product which is purified by recrystallisation from ethyl acetate- hexane to give the title compound as a yellow crystalline solid, m.p. 126-128 °C.
• Nitric acid (1.04 mL of 65%, 15 mmol) is added to a stirred solution of 2-[(2-propenyloxy)- methyl]-5-nitrobenzenamine (3.15 g, 15 mmol) in ethanol (30 mL) at 20°C.
• a solution of cyanamide (0.95 g, 22.5 mmol) in water (1 mL) is then added dropwise to the stirred mixture at 95°C over a period of 60 min.
• the mixture is heated at 95°C for 14 h, with additional aliquots of cyanamide (total 2.2 g, 58 mmol) being added throughout this period and with the acidity being periodically adjusted to pH 3 by the addition of nitric acid (65%).
• Step 1.4 /V-(2-r(2-Propenyloxy)-methvn-5-nitro-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine
• Step 1.5 ⁇ /-(2-Hvdroxymethyl-5-nitro-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine
• Polymethylhydrosiloxane (860 mg), tetrakis(triphenylphoshine)palladium (70 mg) and zinc chloride (2.66 mL of 0.5 M in THF, 1.33 mmol) is added to a stirred solution of ⁇ /- ⁇ 2-[(2- propenyloxy)-methyl]-5-nitro-phenyl ⁇ -4-(3-pyridinyl)-2-pyrimidinamine (2.60 g, 7.2 mmol) in dry THF (60 mL).
• Step 1.6 ⁇ /-(5-Amino-2-hvdroxymethyl-phenyl)-4-(3-pyridinyl)-2-pyrimidinamine
• Example 2 4-IT4-Methyl-4-oxido-1 -piperazinyl)-methyl1-N-(4-methyl-3-rf4-(3-pyridinyl)-2- pyrimidinyll-aminol-phenvD-benzamide 3-Chloroperoxybenzoic acid (Fluka, Buchs, Switzerland; 2.06 g of 55%, 4.27 mmol ) is added to a stirred mixture of 4-[(4-methyl-1-piperazinyl)-methyl]- ⁇ /- ⁇ 4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]-amino]-phenyl ⁇ -benzamide (prepared as described in EP 0 564 409 B1 , Example 21 ; 2.00 g, 4.05 mmol) in dichloromethane (70 mL) at -20°C.
• Example 3 4-r(4-Methyl-1 -piperazinyl)-methvn- ⁇ /-r4-methyl-3-rr4-(1 -oxido-3-pyridinyl)-2- pyrimidinyll-aminol-phenvn-benzamide
• ⁇ -Methylpiperazine (99 mg, 1.0 mmol) is added to a stirred suspension of 4-chloromethyl-/V- [4-methyl-3-[[4-(1 -oxido-3-pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide (220 mg, 0.49 mmol) in ethanol (5 mL). The mixture is then stirred at 100°C for 15 h to give a solution, which is then cooled to RT and treated with ethyl acetate (200 mL).
• Step 3.1 ⁇ /-r4-Methyl-3-fr4-(1-oxido-3-pyridinyl)-2-pyrimidinv ⁇ -aminol-phenyll-benzamide
• ⁇ /-[4-methyl-3-[[4-(3-pyridinyl)- 2-pyrimidinyl]-amino]-phenyl]-benzamide prepared as described in EP 0 564 409 B1 , Example 20
• 4-[(4-methyl-1 -piperazinyl)-methyl]-/V- ⁇ 4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]-amino]-phenyl ⁇ -benzamide afforded the title compound which is purified by column chromatography on silica gel, eluent 10% methanol in dichloromethane, and recrystallised from ethanol to give the title compound as a pale-yellow crystalline
• Step 3.2 4-Methyl- ⁇ /-3-r4-(1-oxido-3-pyridinyl)-2-pyrimidinvn-1.3-benzenediamine Hydrochloric acid (9 mL of 4M) is added to a suspension of ⁇ /-[4-methyl-3-[[4-(1-oxido-3- pyridinyl)-2-pyrimidinyl]-amino]-phenyl]-benzamide (0.43 g, 1.08 mmol) in n-propanol (9 mL) and the resulting mixture is heated at 100°C for 34 h.
• Step 3.3 4-Chloromethyl- ⁇ /-r4-methyl-3-IT4-(1 -oxido-3-pyridinyl)-2-pyrimidinyl -amino]- phenyll-benzamide
• Example 4 4-f(4-Methyl-1 ,4-dioxido-1-piperazinyl)-methyll-/V-(4-methyl-3-rf4-(3-pyridinyl)-2- pyrimidinvn-aminol-phenvD-benzamide
• Tablets containing 100 mg of a compound of formula II, for example one of the compounds of formula II described in the Examples 1-4, are usually prepared in the following composition:
• the active substance is mixed with carrier materials and compressed on a tableting machine (Korsch EKO, punch diameter 10 mm).
• Avicel is microcrystalline cellulose (FMC, Philadelphia, USA).
• PVPPXL is polyvinylpolypyrrolidone, cross-linked (BASF, Germany). Aerosil is silicon dioxide (Degussa, Germany).
• Capsules containing 100 mg of a compound of formula II, for example one of the compounds of formula II described in the Examples 1-4, are usually prepared in the following composition:
• the capsules are prepared by mixing the components and filling the mixture into hard gelatin capsules, size 1.

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